ARF (ADP-ribosylation factor) proteins were first identified by their ability to activate cholera toxin-catalyzed ADP-ribosylation. These GTP- binding proteins, now known to function in intracellular vesicular transport, are active with GTP bound, inactive when it is hydrolyzed to GDP. To activate inactive ARF-GDP, a guanine nucleotide-exchange protein (GEP) is required to promote dissociation of GDP and binding of GTP, which is present in cells at higher concentration. Inhibition of this process by brefeldin A (BFA), a fungal metabolite that interferes with vesicular transport has been reported by several groups. A 700-kDa soluble protein complex containing GEP activity that was inhibited by BFA had been described earlier by Dr. Vaughan's group. Partial purification yielded about 60-kDa BFA-insensitive GDP that enhanced binding of ARF1 and ARF3 (Class I) to Golgi membranes. After purifying a second BFA- insensitive GDP, the possibilities remained that either ARF GDP is not itself a target of BFA, or that there exist BFA-insensitive as well as BFA-sensitive forms of ARF GDP. Attempts to isolate a BFA-sensitive ARF GEP have now been successful. A purified about 700-kDa complex contained a 200-kDa GEP protein that was inhibited by BFA. Like the other purified ARF GEPs, it was apparently active only with class I, preferably myristoylated, ARF proteins. Like GDP dissociation, GTP hydrolysis requires ARF interaction with another protein. An ARF GTPase-activating protein (GAP) purified from rat spleen cytosol appeared to be a homodimer of 50-kDa proteins. The specificity of the purified ARF GAP is clearly much broader than that of ARF GEPs thus far purifed, which seem to interact exclusively with class I ARFs (ARF1 and ARF3) that have been post-translationally modified by N-terminal myristoylation. It seems that the spectrum of the GAP substrates could even include ARL1, a GTP-binding protein of unknown function that is 53% identical in amino acid sequence to ARF1 (181 residues in each). Dramatic effects of specific phospholipids on the activities of the ARF GAP and all of the ARF GEPs have been demonstrated.